1. Field of the Invention
The present invention is relates to proteomics and more specifically involves analyzing polypeptides and peptides using stable isotope labeling techniques coupled with mass spectrometry.
2. Description of Related Art
Proteomics usually involves separating individual proteins using two dimensional gel electrophoresis (2D-PAGE) and comparing stain density. Proteomic analyses using 2D-PAGE can be automated, but only at significant expense requiring automated gel staining and destaining devices, imaging equipment, imaging software, spot cutting robotics, automated in-gel digestion, robotic MALDI plate spotting, and mass spectrometry. Even expensive high throughput 2D-PAGE systems are known to have difficulties with higher molecular weight proteins, membrane proteins, and highly acidic or basic proteins. Despite the high resolution separations of proteins provided by 2D-PAGE, the method still suffers from a limited dynamic range and low abundance proteins are very difficult to detect in the presence of high abundance proteins. Nevertheless, 2D-PAGE has been the state of the art for making quantitative proteomic measurements.
Reversible biotinylation of cysteinyl peptides has been utilized in a method for the rapid identification of components in a protein mixture (Spahr et al., 2000). In a representative method, a protein mixture is digested and the resulting peptide fragment's cysteine residues biotinylated with a cleavable biotinylation reagent (i.e., N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio)propionamide, commonly known to as “biotin-HPDP”). The biotinylated peptides are then isolated using avidin affinity chromatography and then eluted from the avidin by treatment with dithiothreitol (DTT), which cleaves the link between the biotin and peptide fragment releasing the peptide fragment. The released peptide fragment has free sulfhydryl groups that are alkylated by treatment with iodoacetamide. The alkylated peptide fragments are then analyzed by LC/MS/MS to provide proteomic information. The method described above simplifies complex peptide mixtures for proteomic analysis.
Another analytical method involves labeling proteolytic peptides with different stable isotopes depending on the protein source (e.g., control cells versus stimulated cells). Identical peptides labeled with different isotopes have nearly equivalent chemical properties, so pairs of peptides differing only in the label will elute approximately at the same time and exhibit identical ionization efficiency. The first example of this method was the use of whole cell 15N labeling to compare wild type and mutant cell lines. This approach is limited to studies of cultured cells, and the isotope coding involves the incorporation of varying numbers of nitrogen atoms in each peptide, hence varying mass differences from peptide to peptide.
Another approach involves N-terminally labeling proteolytic peptides with isotope-coded nicotinic acid derivatives. This method has a side benefit of directing fragmentation in MS/MS. More recently, whole cell labeling with 13C lysine has been shown to be a simple way to introduce a constant mass shift in tryptic peptides.
In addition to the isotope labeling methods noted above, complex protein mixtures have also been quantitatively analyzed using isotope-coded affinity tags and mass spectrometry (Aebersold et al., 1999). The analysis is based on labeling a protein's cysteine residues with an isotope-coded affinity tag (ICAT) and subsequent analysis of the tagged protein, or fragment thereof, by mass spectrometry. The ICAT reagents employ cysteine-specific chemical reactivity, an isotope coded linker, and a biotin affinity tag, and introduce a constant mass difference for each cysteine present in the peptide. The ICAT reagent includes a reactive functional group having specificity toward sulfhydryl groups, a biotin affinity tag, and an isotope labeled linker covalently linking the sulfhydryl reactive group with the biotin tag. An advantage of this method is that complex tryptic peptide mixtures can be simplified by the selective isolation of peptides containing cysteine, which is one of the least common amino acids, thus approaching the ideal of obtaining a single peptide per protein.
In a representative method, the cysteinyl residues in a reduced protein sample representing one cell state are derivatized with one isotopic form (e.g., light form, no isotope label) of the ICAT and the equivalent groups in a second cell state are derivatized with another isotopic form (i.e., heavy form, isotope labeled). The two samples are then combined, enzymatically cleaved to produce peptide fragments, and the biotin tagged fragments isolated by avidin affinity chromatography. The isolated fragments are then released and analyzed by microLC-MS/MS. The quantity and sequence identity of the proteins from which the fragments are derived are determined by automated multistage mass spectrometry. Despite the utility of the ICAT method described above, the method requires the use of the relatively sophisticated and expensive ICAT reagent. Furthermore, the mass spectra of tagged protein fragments is obscured by high intensity ions related to the reagent.
Despite the advances in protein analysis, there exists a need for rapid, reliable, and efficient methods for analyzing complex protein mixtures. The present invention seeks to fulfill this need and provides further related advantages.